European Review for Medical and Pharmacological Sciences 2019; 23: 4770-4776 LncRNA LUCAT1 promotes growth, migration, and invasion of oral squamous cell carcinoma by upregulating PCNA

Y. KONG1, Y. FENG2, Y.-Y. XIAO3, S.-C. LIU4, X.-G. LI5, Q.-L. YANG1, W.-H. CHU1, J.-G. LIU6

1Department of Pedodontics, Affiliated Stomatological Hospital of Jiamusi University in Heilongjiang Province, Jiamusi, China 2Department of Conservative Dentistry and Endodontics, Affiliated Stomatological Hospital of Jiamusi University in Heilongjiang Province, Jiamusi, China 3Department of Repair Section, Affiliated Stomatological Hospital of Jiamusi University in Heilongjiang Province, Jiamusi, China 4Department of Orthopedics, First Affiliated Hospital of Jiamusi University in Heilongjiang Province, Jiamusi, China 5Department of Orthodontic, Affiliated Stomatological Hospital of Jiamusi University in Heilongjiang Province, Jiamusi, China 6Department of Graduate Student, Jiamusi University, Jiamusi, China

Yu Kong and Yao Feng contributed equally to this work

Abstract. – OBJECTIVE: Recent studies have Key Words: revealed the important role of long noncoding Long noncoding RNA, LUCAT1, Oral squamous cell RNAs (lncRNAs) in the progression of tumori- carcinoma, PCNA. genesis. This study aims to identify how lncRNA LUCAT1 functions in the progression of OSCC (oral squamous cell carcinoma). PATIENTS AND METHODS: Relative expres- Introduction sion of lncRNA LUCAT1 in both OSCC cells and 50 paired tissue samples was detected by quan- Oral cancer is the sixth most prevalent cancer in titative Real-time polymerase chain reaction the world, and it ranks the third most common in (qRT-PCR). Moreover, biological function of LU- developing countries1. Oral squamous cell carci- CAT1 in OSCC was identified by performing tran- swell assay, wound healing assay and prolifera- noma (OSCC) is the main subtype of oral cancer, tion assay in vitro. The underlying mechanism contributing to 3% of the newly diagnosed clinical 2 was explored by qRT-PCR and Western blot. cancer cases . Although the rapid development of di- RESULTS: LUCAT1 expression was remark- agnosis and treatment methods for OSCC has been ably downregulated in OSCC tissues when com- achieved2, the overall 5-year survival rate of OSCC pared with that in adjacent normal samples. patients still remains lower than 50%. Therefore, it Moreover, proliferation, invasion and migration is essential and urgent to find out novel molecular of OSCC cells were inhibited after knockdown of LUCAT1 in vitro. Knockdown of LUCAT1 down- markers to improve the treatment efficacy of OSCC. regulated PCNA in OSCC cells at mRNA and pro- Long non-coding RNA (lncRNA) is a cluster of tein level in vitro. Besides, PCNA expression in non-coding RNA transcripts with over 200 nucle- OSCC tissues was positively correlated with the otides (nt) in length and it lacks the ability of en- expression of LUCAT1. coding proteins. Evidence shows that lncRNAs act CONCLUSIONS: Knockdown of LUCAT1 could as vital regulators in many biological behaviors, inhibit migration, invasion and proliferation ca- pacities of OSCC cells through downregulating including carcinogenesis, apoptosis, proliferation PCNA, which may offer a new therapeutic inter- and metastasis in cancer. For example, lncRNA ventionRETRACTED for OSCC patients. OR3A4 is upregulated in breast cancer, which may Corresponding Author: Xiaoguang Li, MM; e-mail: [email protected] 4770 Jiguang Liu, MM; e-mail: [email protected] Role of lncRNA LINC00052 in cervical cancer be a novel prognostic marker and therapeutic tar- pcDNA3.1 into OSCC cells with Lipofectamine get3. Activated by H3K27, lncRNA CCAT1 pro- 2000 (Invitrogen, Carlsbad, CA, USA). Transfection motes cell proliferation and migration in esophageal efficacy was verified by qRT-PCR. squamous cell carcinoma through regulating SPRY4 and HOXB134. LncRNA MALAT1 stimulates the RNA Extraction and qRT-PCR cell migration and invasion in hepatocellular carci- Total RNA from tissues and cells were ex- noma by sponging miR-204 to upregulate SIRT15. tracted using TRIzol reagent (Invitrogen, Carls- By suppressing p53 signal pathway, lncRNA ROR bad, CA, USA). The total RNA was reversely facilitates cell proliferation, invasion and chemore- transcribed to cDNA through reverse Transcrip- sistance in nasopharyngeal carcinoma6. Moreover, tion Kit (TaKaRa Biotechnology Co., Ltd., Da- knockdown of lncRNA CRNDE-h is reported to be lian, China). Thermocycling conditions were associated with poor prognosis of colorectal cancer, as follows: 95°C for 30 s, followed by 40 cycles which might be used as a novel serum-based bio- at 95°C for 5 s, and 60°C for 35 s. Primers used marker for diagnosis of colorectal cancer7. However, for qRT-PCR were as follows: LUCAT1, forward the potential influence of lncRNA LUCAT1 on the 5′-CCTATCCCTTTCTCTAAGAA-3′ and reverse development of OSCC remains unexplored. In the 5-ACTTCTGCAAAAACGTGCTG-3′; glyceral- present study, it is revealed that the expression level dehyde 3-phosphate dehydrogenase (GAPDH), of lncRNA LUCAT1 was remarkably upregulated in forward 5′-CCAAAATCAGATGGGGCAAT- OSCC samples. Moreover, functional experiments GCTGG-3′ and reverse 5′-TGATGGCATGGACT- revealed that knockdown of LUCAT1 depressed GTGGTCATTCA-3′. The thermal cycle was as cell proliferation, invasion and migration capacities follows: 95°C for 30 s, followed by 40 cycles at of OSCC cells. Furthermore, we discovered that ln- 95°C for 5 s and 60°C for 35 s. 2-ΔΔCt method was cRNA LUCAT1 mediated the progression of OSCC utilized for calculating relative expression. by promoting PCNA. Western Blot Patients and Methods Total proteins were collected from cells via radioimmunoprecipitation assay (RIPA) buffer Tissue Specimens (Beyotime, Shanghai, China) and then quantified Paired OSCC tissues and adjacent non-tumor by bicinchoninic acid (BCA) protein quantifica- tissues (≥ 5 cm away from the tumor site) were tion kit (Pierce, Rockford, IL, USA). The target sequentially resected from 50 OSCC patients proteins were separated by sodium dodecyl sul- during July 2015 and December 2017 in the Sec- phate-polyacrylamide gel electrophoresis (SDS- ond Affiliated Hospital of Jiamusi University in PAGE). Membranes were incubated with primary Heilongjiang Province. The Ethics Committee antibodies after loading on the polyvinylidene of Affiliated Stomatological Hospital of Jiamusi difluoride (PVDF) membrane (Millipore, Billeri- University in Heilongjiang Province approved ca, MA, USA). Then rabbit anti-GAPDH and rab- this study protocol, and all participants provided bit anti-PCNA were used for incubation. Pierce written informed consents. electrochemiluminescence (ECL) was utilized for visualizing Western blotting substrate immuno- OSCC Cells and Cell Transfection reactive bands (Santa Cruz Biotechnology, Santa Human OSCC cell lines Tca8113, TSCCA, CAL- Cruz, CA, USA). 27, SCC-9 and normal cervical epithelium cell line NHOK were provided by Chinese Academy of Sci- Cell Proliferation Assay ence (Shanghai, China). Cells were cultured in Ros- Cell proliferation of treated cells in 96-well well Park Memorial Institute-1640 (RPMI-1640; plates was monitored every 24 h by cell count- HyClone, South Logan, UT, USA) containing 10% ing kit-8 (CCK-8) assay following the instructions fetal bovine serum (FBS; Invitrogen Life Technolo- (Dojindo Molecular Technologies, Inc., Kumamo- gies, Gaithersburg, MD, USA), and 1% penicillin in to, Japan). Spectrophotometer (Thermo Scientific, a 5% CO2 incubator at 37°C. Lentivirus expressing Waltham, MA, USA) was utilized to measure the short-hairpin RNA (shRNA) directed against LU- absorbance at 450 nm. CAT1 were provided by GenePharma (Shanghai, China). cDNA encoding LUCAT1 was amplified Wound Healing Assay andRETRACTED inserted into pcDNA3.1 (Invitrogen, Carlsbad, Cells seeded into 6-well plates were cultured CA, USA). Next, we transfected the synthesized in RPMI-1640 medium overnight. After scratch-

4771 Y. Kong, Y. Feng, Y.-Y. Xiao, S.-C. Liu, X.-G. Li, Q.-L. Yang, W.-H. Chu, J.-G. Liu ing with a plastic tip, cells were cultured in se- LUCAT1 was significantly upregulated in tumor rum-free RPMI-1640. Wound closure was detect- tissues than that in adjacent tissues (Figure 1A). ed at the appointed time points. Each assay was When compared with the expression in normal hu- independently repeated for three times. man oral keratinocyte NHOK, LUCAT1 level was significantly higher in OSCC cells (Figure 1B). Transwell Assay Twenty-four hours after transfection, 2×105 cells Knockdown of LUCAT1 Repressed Cell in 100 µL of serum-free RPMI-1640 were applied Proliferation In Vitro in the top side of an 8-μm Transwell chamber According to LUCAT1 expression in cancer (Corning, Corning, NY, USA) pre-coated with 50 cells, we chose SCC-9 cells for establishing the µg Matrigel (BD Biosciences, Franklin Lakes, NJ, LUCAT1 knockdown model. The LUCAT1 lenti- USA). RPMI-1640 containing 20% fetal bovine virus (LUCAT1) and the empty vector were syn- serum (FBS) was added to the bottom chamber. thetized, and their transfection efficacy in SCC-9 After 24 h, cells were fixed in methanol for 30 min cells was determined by qRT-PCR (Figure 2A). and stained by hematoxylin for 20 min. An invert- Furthermore, CCK-8 assay showed that prolifer- ed microscope (×20) was utilized for counting mi- ation of OSCC cells was inhibited after LUCAT1 grated and invaded cells in three random fields. knockdown (Figure 2B).

Statistical Analysis Knockdown of LUCAT1 Repressed Cell Statistical analysis was conducted by Statis- Migration and Invasion In Vitro tical Product and Service Solutions (SPSS) 18.0 Wound healing assay revealed that knockdown (SPSS Inc., Chicago, IL, USA). Chi-square test of LUCAT1 inhibited OSCC cell migration (Fig- and Student t-test were performed to analyze cat- ure 3A). Furthermore, transwell assay showed egorical data or measurement data, respectively. that invasion of OSCC cells was inhibited after Kaplan-Meier curve was introduced for surviv- LUCAT1 knockdown (Figure 3B). al analysis. Data were presented by mean ± SD (Standard Deviation). It was considered statisti- LUCAT1 Promoted OSCC Tumorigenesis cally significant when p<0.05. via PCNA QRT-PCR results further demonstrated that the mRNA expression of PCNA was downregulated Results in OSCC cells transfected with LUCAT1 shR- NA (Figure 4A). Western blot analysis obtained Expression Level of LUCAT1 in Tissues the same trend at the protein level (Figure 4B). and Cells of OSCC To explore the interaction between LUCAT1 and First, qRT-PCR was conducted for detecting PCNA, the expression level of PCNA was detect- LUCAT1 expression in 50-paired OSCC tissues. ed in OSCC tissues. PCNA expression was mark-

Figure 1. Expression levels of LUCAT1 were increased in OSCC tissues and cell lines. A, LUCAT1 expression was signifi- cantly increased in the OSCC tissues compared with adjacent tissues. B, Expression levels of LUCAT1 relative to GAPDH wereRETRACTED determined in the human OSCC cell lines and normal human oral keratinocyte (NHOK) by qRT-PCR. Data are presented as the mean ± standard error of the mean. *p<0.05.

4772 Role of lncRNA LINC00052 in cervical cancer

Figure 2. Knockdown of LUCAT1 inhibited OSCC cell proliferation. A, LUCAT1 expression in OSCC cells transduced with empty vector or LUCAT1 lentivirus (sh-LUCAT1) was detected by qRT-PCR. GAPDH was used as an internal control. B, CCK8 assay showed that knockdown of LUCAT1 significantly repressed cell proliferation in SCC-9 OSCC cells. The results represent the average of three independent experiments (mean ± standard error of the mean). *p<0.05.

edly upregulated in OSCC tissues when com- cRNA UCA1 accelerates cell proliferation and pared with that in adjacent tissues (Figure 4C). cisplatin-resistance in oral squamous cell car- The results of linear correlation analysis showed cinoma9. LncRNA MALAT1, which may be an that in OSCC tissues, the expression of PCNA important prognostic biomarker and therapeutic was positively correlated to LUCAT1 expression target for OSCC, promotes the progression of (Figure 4D). OSCC by inducing epithelial-mesenchymal tran- sition10. LncRNA FTH1P3 enhances the devel- opment of OSCC by modulating the expression Discussion of fizzled 11 5 . In addition, lncRNAAC132217.4 promotes cell metastasis in OSCC by regulating Evidence demonstrated that lncRNAs par- IGF2 expression, which is further modulated ticipate in diverse biological behaviors in the by KLF812. Lung cancer associated transcript 1 progression of OSCC, including cell growth, (LUCAT1) was firstly found in the airway ep- metastasis and invasion. For instance, lncRNA ithelium of cigarette smokers, which is a clus- HOTAIR facilitates cell invasion and metasta- ter of transcripts located on chromosome 513. sis in OSCC by indirectly recruiting EZH2 and Recently, lncRNA LUCAT1 is widely studied depressing E-cadherin8. By modulating SF1 and for its important role in the progression of tu- suppressing expression level of miR-184, ln- morigenesis. For instance, through inhibiting

Figure 3. Knockdown of LUCAT1 repressed OSCC cell migration and invasion. A, Wound healing assay showed that the migrated length of cells in LUCAT1 lentivirus group was significantly decreased compared with empty control group in SCC- 9 OSCCRETRACTED cells. B, Transwell assay showed that knockdown of LUCAT1 significantly repressed cell invasion in SCC-9 OSCC cells. The results represent the average of three independent experiments (mean ± standard error of the mean). *p<0.05.

4773 Y. Kong, Y. Feng, Y.-Y. Xiao, S.-C. Liu, X.-G. Li, Q.-L. Yang, W.-H. Chu, J.-G. Liu

Figure 4. Interaction between PCNA and LUCAT1 in OSCC. A, The RNA expression level of PCNA in LUCAT1 cells was significantly decreased compared with empty control cells in SCC-9 cells.B, Protein expression of PCNA was decreased after knockdown of LUCAT1 in SCC-9 cells. C, PCNA was significantly upregulated in OSCC tissues compared with adjacent tissues. D, The linear correlation between the expression level of PCNA and LUCAT1 in OSCC tissues. The results represent the average of three independent experiments. Data are presented as the mean ± standard error of the mean. *p<0.05. the expressions of p21 and p57, lncRNA LUC- and cells. Furthermore, LUCAT1 knockdown AT1 knockdown promotes cell proliferation in inhibited the abilities of cell growth, migration human non-small lung cancer14. Through regu- and invasion. These data indicated that LUC- lating the stability of DNMT1 and depressing AT1 functioned as an oncogene and promoted the expressions of tumor suppressors, lncRNA the tumorigenesis of OSCC. Proliferating cell LUCAT1 promotes the tumorigenesis of esoph- nuclear antigen (PCNA) is found in cancer cells ageal squamous cell carcinoma formation and and proliferating cells. Numerous researches tumor metastasis15. After knockdown of ln- have confirmed that PCNA is directly correlat- cRNA LUCAT1, cell viability and invasion are ed to the prognosis, tumor stage and tumor dif- depressed in glioma through regulating miR- ferentiation. For instance, the positive expres- 375 expression16. LncRNA LUCAT1 enhances sion rate of PCNA was 73% in breast cancer, malignancy of ovarian cancer and promotes indicating the important clinical significance its progression through regulating miR-612/ in evaluating the prognosis of breast cancer19. HOXA13 pathway17. Moreover, the expression The expression level of PCNA is significantly of lncRNA LUCAT1 is negatively correlated higher in colorectal cancer, especially in those to the prognosis of clear cell renal cell carci- with liver metastasis. This may help to evalu- noma and positively related to malignancy of ate liver metastasis in patients with colorectal the RETRACTEDtumor18. In the present study, LUCAT1 was cancer20. Overexpression of PCNA relieves the found to be upregulated in both OSCC tissues repression of miR-363-3p on cell proliferation

4774 Role of lncRNA LINC00052 in cervical cancer and colony formation in lung adenocarcinoma21. the migration and invasion of hepatocellular carci- In addition, upregulation of PCNA is closely re- noma by sponging miR-204 and releasing SIRT1. lated to poor prognosis of patients with osteo- Tumour Biol 2017; 39: 1393371529. sarcoma, which may be a potential biomarker22. 6) Li L, Gu M, You B, Shi S, Shan Y, Bao L, You Y. Long non-coding RNA ROR promotes proliferation, mi- In our study, PCNA was downregulated after gration and chemoresistance of nasopharyngeal LUCAT1 knockdown in vitro. PCNA was re- carcinoma. Cancer Sci 2016; 107: 1215-1222. markably upregulated in OSCC samples when 7) Liu T, Zhang X, Gao S, Jing F, Yang Y, Du L, Zheng G, compared with that in adjacent tissues. A posi- Li P, Li C, Wang C. Exosomal long noncoding RNA tive correlation was discovered between PCNA CRNDE-h as a novel serum-based biomarker for and LUCAT1 expression in OSCC tissues. The diagnosis and prognosis of colorectal cancer. On- above results revealed that LUCAT1 mediated cotarget 2016; 7: 85551-85563. OSCC progression via PCNA. 8) Wu Y, Zhang L, Zhang L, Wang Y, Li H, Ren X, Wei F, Yu W, Liu T, Wang X, Zhou X, Yu J, Hao X. Long non-coding RNA HOTAIR promotes tumor cell invasion and metastasis by recruiting EZH2 and Conclusions repressing E-cadherin in oral squamous cell car- cinoma. Int J Oncol 2015; 46: 2586-2594. Our research suggests a new biomarker in the 9) Fang Z, Zhao J, Xie W, Sun Q, Wang H, Qiao B. development of OSCC. LncRNA LUCAT1 pro- LncRNA UCA1 promotes proliferation and cispla- motes OSCC carcinogenesis via regulating PCNA tin resistance of oral squamous cell carcinoma by sunppressing miR-184 expression. Cancer Med and it may serve as a promising marker for OSCC 2017; 6: 2897-2908. in the future. 10) Zhou X, Liu S, Cai G, Kong L, Zhang T, Ren Y, Wu Y, Mei M, Zhang L, Wang X. Long non coding RNA MALAT1 promotes tumor growth and metastasis by inducing epithelial-mesenchymal transition in oral squamous Conflict of Interests cell carcinoma. Sci Rep 2015; 5: 15972. The Authors declare that they have no conflict of interests. 11) Zhang CZ. Long non-coding RNA FTH1P3 facilitates oral squamous cell carcinoma progression by acting as a molecular sponge of miR-224-5p to modulate Funding Acknowledgements fizzled 5 expression. Gene 2017; 607: 47-55. Li X, Ma C, Zhang L, Li N, Zhang X, He J, He R, Shao This work was supported by Scientific Research Project 12) M, Wang J, Kang L, Han C. of Heilongjiang Health and Family Planning Commission LncRNAAC132217.4, a (2017-416); Key Project of Science and Technology in Ji- KLF8-regulated long non-coding RNA, facilitates oral amusi University (12Z1201509). squamous cell carcinoma metastasis by upregulat- ing IGF2 expression. Cancer Lett 2017; 407: 45-56. 13) Thai P, Statt S, Chen CH, Liang E, Campbell C, Wu R. Characterization of a novel long noncoding RNA, References SCAL1, induced by cigarette smoke and elevated in lung cancer cell lines. Am J Respir Cell Mol Biol 2013; 49: 204-211. 1) Liu LM. Inhibitory effects of int-2 gene on the inva- 14) Sun Y, Jin SD, Zhu Q, Han L, Feng J, Lu XY, Wang W, sion and metastasis of oral cancer cells. Eur Rev Wang F, Guo RH. Long non-coding RNA LUCAT1 Med Pharmacol Sci 2017; 21: 5677-5682. is associated with poor prognosis in human non- 2) Brocklehurst PR, Baker SR, Speight PM. Oral cancer small lung cancer and regulates cell proliferation screening: what have we learnt and what is there via epigenetically repressing p21 and p57 expres- still to achieve? Future Oncol 2010; 6: 299-304. sion. Oncotarget 2017; 8: 28297-28311. Yoon JH, You BH, Park CH, Kim YJ, Nam JW, Lee SK. 3) Liu G, Hu X, Zhou G. Long non-coding RNA 15) OR3A4 promotes proliferation and migration in The long noncoding RNA LUCAT1 promotes tum- breast cancer. Biomed Pharmacother 2017; 96: origenesis by controlling ubiquitination and stability 426-433. of DNA methyltransferase 1 in esophageal squa- mous cell carcinoma. Cancer Lett 2018; 417: 47-57. Zhang E, Han L, Yin D, He X, Hong L, Si X, Qiu M, Xu 4) 16) Gao YS, Liu XZ, Zhang YG, Liu XJ, Li LZ. Knock- T, De W, Xu L, Shu Y, Chen J. H3K27 acetylation ac- down of long noncoding RNA LUCAT1 inhibits cell tivated-long non-coding RNA CCAT1 affects cell viability and invasion by regulating miR-375 in gli- proliferation and migration by regulating SPRY4 oma. Oncol Res 2018; 26: 307-313. and HOXB13 expression in esophageal squa- 17) Yu H, Xu Y, Zhang D, Liu G. Long noncoding RNA mous cell carcinoma. Nucleic Acids Res 2017; 45: LUCAT1 promotes malignancy of ovarian cancer 3086-3101. through regulation of miR-612/HOXA13 path- 5) RETRACTEDHou Z, Xu X, Zhou L, Fu X, Tao S, Zhou J, Tan D, Liu way. Biochem Biophys Res Commun 2018; 503: S. The long non-coding RNA MALAT1 promotes 2095-2100.

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